scholarly journals Thidiazuron, a phenyl-urea cytokinin, inhibits ergosterol synthesis and attenuates biofilm formation of Candida albicans

2021 ◽  
Author(s):  
Harikrishnan Pandurangan ◽  
Balamani Arayambath ◽  
Vijay Karthik Jayaraman ◽  
Kanimozhi Ekambaram ◽  
Emad A Ahmed ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Reduction Assay ◽  
Ergosterol Synthesis ◽  
Inhibitory Potential ◽  
Anti Fungal ◽  
Time Kill

Abstract Candida albicans is a commensal human fungal pathogen that colonizes and develops dental biofilm which cause Oral candidiosis. This study investigates the effects of a new molecule Thidiazuron against the growth and biofilm formation properties of C. albicans. This study applied computational and in vitro approaches such as broth microdilution, SEM, time-kill dynamics, crystal violet assay, XTT reduction assay, ergosterol quantification and quantitative RT PCR analysis of gene expression to validate the growth and biofilm inhibitory potential of thidiazuron against C. albicans. Preliminary molecular docking study revealed potential interaction between thidiazuron and amino acids residues of CYP51. Further in vitro anti-fungal susceptibility test, SEM and time kill analysis revealed anti-fungal potency of thidiazuron in dose and time dependent passion. Crystal violet staining, XTT reduction assay and Acridine Orange staining visually confirmed biofilm inhibitory potential of thidiazuron. Gene expression study shows that thidiazuron treatment down regulated the expression of genes involved in ergosterol synthesis, cell adhesion and hyphae development in C. albicans. This study identified thidiazuron as CYP51 inhibitor and a new antibiofilm agent against C. albicans.

scholarly journals A modified crystal violet assay is comparable to XTT reduction assay for quantification of biofilm formation by Candida albicans

2020 ◽  
Author(s):  
Yue Qu ◽  
Shoufeng Yang ◽  
Zhangzhang Chen ◽  
Feifei Su
Keyword(s):  
Candida Albicans ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Quantitative Methods ◽  
Standard Procedure ◽  
Short Term ◽  
Biofilm Growth ◽  
Human Fungal Pathogen ◽  
Crystal Violet Assay ◽  
Reduction Assay

Abstract Background: The ability of the human fungal pathogen Candida albicans to form biofilms, for example on indwelling medical devices, is a major pathogenic mechanism and has been the focus of intense studies in the fungal pathogenesis field. A key research tool used is the quantitative methods for measuring biofilm formation of C. albicans. Objective: We sought to optimize the conventional crystal violet (CV) staining assay for quantification of biofilm formation by C. albicans and evaluate its performance. Methods: Individual modifications included (i) submerge-washing of microplates to remove non-adherent cells, (ii) heat-fixation, (iii) short-term staining for 3 min, (iv) submerge-washing to remove unbound CV dye, and (v) short-term destaining for 15 min were compared with the standard procedure, and those were superior were incorporated. Performance analysis was carried out for the modified CV assay, in comparison to the conventional CV assay and the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide] reduction assay. Results: The modified CV assay demonstrated several advantages in quantitative assessment of biofilm formation of C. albicans over the conventional CV assay, including greater accuracy and reproducibility, shorter experimental time and reduced labor intensity, and was at least comparable to the XTT reduction assay.Conclusion: The modified CV method can be used as an alternative to the XTT reduction assay in quantification of biofilm growth by C. albicans.

A role of Candida albicans CDC4 in the negative regulation of biofilm formation

2015 ◽  
Vol 61 (4) ◽  
pp. 247-255 ◽  
Author(s):  
Tzu-Ling Tseng ◽  
Wei-Chung Lai ◽  
Tai-Lin Lee ◽  
Wan Hua Hsu ◽  
Yu Wen Sun ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Candida Albicans ◽  
Biofilm Formation ◽  
Negative Regulation ◽  
Null Mutant ◽  
Auxotrophic Strain ◽  
Reduction Assay ◽  
Mutant Strains

The CDC4 gene is nonessential in Candida albicans and plays a role in suppressing filamentous growth, in contrast to its homologues, which are involved in the G1–S transition of the cell cycle. While characterizing the function of C. albicans CDC4 (CaCDC4), we found that the loss of CaCDC4 resulted in a reduction in cell flocculation, indicating a possible role for CaCDC4 in biofilm formation. To elucidate the role of CaCDC4 in biofilm formation, Cacdc4 null mutant strains were constructed by using the mini-Ura-blaster method. To create a CaCDC4 rescued strain, the plasmid p6HF-ACT1p-CaCDC4 capable of constitutively expressing CaCDC4 was introduced into the Cacdc4 homozygous null mutant. To determine the biofilm formation ability, an in vitro XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-5-carboxanilide) reduction assay was used. Compared with the parental auxotrophic strain BWP17, the Cacdc4 homozygous null mutant was able to enhance biofilm formation significantly. This enhancement of biofilm formation in the Cacdc4 homozygous null mutant could be reversed by constitutively expressing CaCDC4. We conclude that CaCDC4 has a role in suppressing biofilm formation in C. albicans.

scholarly journals ALS3 Expression as an Indicator for Candida albicans Biofilm Formation and Drug Resistance

2021 ◽  
Vol 12 ◽  
Author(s):  
Keke Deng ◽  
Wei Jiang ◽  
Yanyu Jiang ◽  
Qi Deng ◽  
Jinzhong Cao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Biofilm Formation ◽  
Antifungal Agents ◽  
Early Prevention ◽  
Therapy Failure ◽  
Prevention And Treatment ◽  
Selection Of ◽  
Prevention And Therapy

Resistance caused by the formation of the Candida albicans (C. albicans) biofilm is one of the main reasons for antifungal therapy failure. Thus, it is important to find indicators that predict C. albicans biofilm formation to provide evidence for the early prevention and treatment of the C. albicans biofilms. In this study, C. albicans samples were selected from C. albicans septicemia that were sensitive to common antifungal agents. It was found that the agglutinin-like sequence 3 (ALS3) gene was differentially expressed in free, antifungal, drug-sensitive C. albicans. The average ALS3 gene expression was higher in the C. albicans strains with biofilm formation than that in the C. albicans strains without biofilm formation. Then, it was further confirmed that the rate of biofilm formation was higher in the high ALS3 gene expression group than that in the low ALS3 gene expression group. It was found that C. albicans with biofilm formation was more resistant to fluconazole, voriconazole, and itraconazole. However, it maintained its sensitivity to caspofungin and micafungin in vitro and in mice. Further experiments regarding the prevention of C. albicans biofilm formation were performed in mice, in which only caspofungin and micafungin prevented C. albicans biofilm formation. These results suggest that the expression level of ALS3 in C. albicans may be used as an indicator to determine whether C. albicans will form biofilms. The results also show that the biofilm formation of C. albicans remained sensitive to caspofungin and micafungin, which may help to guide the selection of clinical antifungal agents for prevention and therapy.

scholarly journals Monitoring ALS1 and ALS3 Gene Expression During In Vitro Candida albicans Biofilm Formation Under Continuous Flow Conditions

2008 ◽  
Vol 167 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Heleen Nailis ◽  
Roosmarijn Vandenbroucke ◽  
Kelly Tilleman ◽  
Dieter Deforce ◽  
Hans Nelis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Continuous Flow ◽  
Biofilm Formation ◽  
Flow Conditions

scholarly journals The Synergistic Effect of Azoles and Fluoxetine against Resistant Candida albicans Strains Is Attributed to Attenuating Fungal Virulence

2016 ◽  
Vol 60 (10) ◽  
pp. 6179-6188 ◽  
Author(s):  
Wenrui Gu ◽  
Dongmei Guo ◽  
Liuping Zhang ◽  
Dongmei Xu ◽  
Shujuan Sun
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Synergistic Effects ◽  
Mechanism Study ◽  
Content Type ◽  
Expression Levels ◽  
Time Kill ◽  
Gene Expression Levels

ABSTRACTThis study evaluated the synergistic effects of the selective serotonin reuptake inhibitor, fluoxetine, in combination with azoles againstCandida albicansbothin vitroandin vivoand explored the underlying mechanism. MICs, sessile MICs, and time-kill curves were determined for resistantC. albicans.Galleria mellonellawas used as a nonvertebrate model for determining the efficacy of the drug combinations againstC. albicansin vivo. For the mechanism study, gene expression levels of theSAPgene family were determined by reverse transcription (RT)-PCR, and extracellular phospholipase activities were detectedin vitroby the egg yolk agar method. The combinations resulted in synergistic activity againstC. albicansstrains, but the same effect was not found for the non-albicans Candidastrains. For the biofilms formed over 4, 8, and 12 h, synergism was seen for the combination of fluconazole and fluoxetine. In addition, the time-kill curves confirmed the synergism dynamically. The results of theG. mellonellastudies agreed with thein vitroanalysis. In the mechanism study, we observed that fluconazole plus fluoxetine caused downregulation of the gene expression levels ofSAP1toSAP4and weakened the extracellular phospholipase activities of resistantC. albicans. The combinations of azoles and fluoxetine showed synergistic effects against resistantC. albicansmay diminish the virulence properties ofC. albicans.

scholarly journals Multi-Omics Reveals the Inhibition of Lactiplantibacillus plantarum CCFM8724 in Streptococcus mutans-Candida albicans Mixed-Species Biofilms

2021 ◽  
Vol 9 (11) ◽  
pp. 2368
Author(s):  
Qiuxiang Zhang ◽  
Jiaxun Li ◽  
Wenwei Lu ◽  
Jianxin Zhao ◽  
Hao Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Dental Caries ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Metabolic Reprogramming ◽  
Mixed Species ◽  
Carbohydrate Utilization

Lactiplantibacillus plantarum CCFM8724 is a probiotic with the potential to prevent dental caries in vitro and in vivo. To explore the effects of this probiotic at inhibiting Streptococcus mutans-Candida albicans mixed-species biofilm and preventing dental caries, multi-omics, including metabolomics and transcriptomics, was used to investigate the regulation of small-molecule metabolism during biofilm formation and the gene expression in the mixed-species biofilm. Metabolomic analysis revealed that some carbohydrates related to biofilm formation, such as sucrose, was detected at lower levels due to the treatment with the L. plantarum supernatant. Some sugar alcohols, such as xylitol and sorbitol, were detected at higher levels, which may have inhibited the growth of S. mutans. In transcriptomic analysis, the expression of the virulence genes of C. albicans, such as those that code agglutinin-like sequence (Als) proteins, was affected. In addition, metabolomics coupled with a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and RNA-seq revealed that the L. plantarum supernatant had an active role in sugar metabolism during the formation of the S. mutans-C. albicans mixed-species biofilm, and the L. plantarum supernatant was also related to carbohydrate utilization, glucan biosynthesis, and mycelium formation. Hence, L. plantarum CCFM8724 decreased the mixed-species biofilm mass from the perspective of gene expression and metabolic reprogramming. Our results provide a rationale for evaluating L. plantarum CCFM8724 as a potential oral probiotic for inhibiting cariogenic pathogen biofilm formation and improving dental caries.

scholarly journals Synergistic activity of azoles with amiodarone against clinically resistant Candida albicans tested by chequerboard and time–kill methods

2008 ◽  
Vol 57 (4) ◽  
pp. 457-462 ◽  
Author(s):  
Qiongjie Guo ◽  
Shujuan Sun ◽  
Jinlong Yu ◽  
Yan Li ◽  
Lili Cao
Keyword(s):  
Candida Albicans ◽  
Inhibitory Concentration ◽  
Systemic Disease ◽  
Synergistic Activity ◽  
Synergistic Interactions ◽  
Colony Counting ◽  
Reduction Assay ◽  
Time Kill ◽  
Concentration Indices

Candida albicans is the most common candidal pathogen, causing serious systemic disease in immunocompromised patients. Azoles are widely applied and largely effective; however, they are generally fungistatic and clinically resistant isolates are emerging increasingly. The present study provided in vitro evidence using a chequerboard technique that amiodarone is strongly synergistic with azoles against resistant C. albicans, with mean fractional inhibitory concentration indices of 0.01 and high-percentage synergistic interactions of 1250 %. A time–kill study performed by both colony counting and a colorimetric reduction assay confirmed the synergistic interaction, with a ≥2 log10 decrease in c.f.u. ml−1 compared with the corresponding azoles alone. These results suggest the possibility of supplementing azoles with amiodarone to treat resistant C. albicans infections.

scholarly journals Regulatory Role of Glycerol in Candida albicans Biofilm Formation

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Jigar V. Desai ◽  
Vincent M. Bruno ◽  
Shantanu Ganguly ◽  
Ronald J. Stamper ◽  
Kaitlin F. Mitchell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Cell Surface ◽  
Biofilm Formation ◽  
Regulatory Role ◽  
Content Type ◽  
Upregulated Genes ◽  
Implanted Devices

ABSTRACTBiofilm formation byCandida albicanson medically implanted devices poses a significant clinical challenge. Here, we compared biofilm-associated gene expression in two clinicalC. albicansisolates, SC5314 and WO-1, to identify shared gene regulatory responses that may be functionally relevant. Among the 62 genes most highly expressed in biofilms relative to planktonic (suspension-grown) cells, we were able to recover insertion mutations in 25 genes. Twenty mutants had altered biofilm-related properties, including cell substrate adherence, cell-cell signaling, and azole susceptibility. We focused on one of the most highly upregulated genes in our biofilm proles,RHR2, which specifies the glycerol biosynthetic enzyme glycerol-3-phosphatase. Glycerol is 5-fold-more abundant in biofilm cells than in planktonic cells, and anrhr2Δ/Δ strain accumulates 2-fold-less biofilm glycerol than does the wild type. Underin vitroconditions, therhr2Δ/Δ mutant has reduced biofilm biomass and reduced adherence to silicone. Therhr2Δ/Δ mutant is also severely defective in biofilm formationin vivoin a rat catheter infection model. Expression profiling indicates that therhr2Δ/Δ mutant has reduced expression of cell surface adhesin genesALS1,ALS3, andHWP1, as well as many other biofilm-upregulated genes. Reduced adhesin expression may be the cause of therhr2Δ/Δ mutant biofilm defect, because overexpression ofALS1,ALS3, orHWP1restores biofilm formation ability to the mutantin vitroandin vivo. Our findings indicate that internal glycerol has a regulatory role in biofilm gene expression and that adhesin genes are among the main functional Rhr2-regulated genes.IMPORTANCECandida albicansis a major fungal pathogen, and infection can arise from the therapeutically intractable biofilms that it forms on medically implanted devices. It stands to reason that genes whose expression is induced during biofilm growth will function in the process, and our analysis of 25 such genes confirms that expectation. One gene is involved in synthesis of glycerol, a small metabolite that we find is abundant in biofilm cells. The impact of glycerol on biofilm formation is regulatory, not solely metabolic, because it is required for expression of numerous biofilm-associated genes. Restoration of expression of three of these genes that specify cell surface adhesins enables the glycerol-synthetic mutant to create a biofilm. Our findings emphasize the significance of metabolic pathways as therapeutic targets, because their disruption can have both physiological and regulatory consequences.

scholarly journals In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

2011 ◽  
Vol 56 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Marisa H. Miceli ◽  
Stella M. Bernardo ◽  
T. S. Neil Ku ◽  
Carla Walraven ◽  
Samuel A. Lee
Keyword(s):  
Candida Albicans ◽  
Metabolic Activity ◽  
Biofilm Formation ◽  
High Dose ◽  
Dependent Manner ◽  
Planktonic Cells ◽  
Content Type ◽  
Heparin Sodium ◽  
The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

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